Patent AT-E353954-T1 (AT)
Inventor: WINQVIST OLA
IPC: A61K39/00
Filed: 2002-10-11
Granted: 2007-03-15
Abstract: Treating and/or preventing (M1) the recurrence of cancer, comprises providing lymphocytes obtained from sentinel lymph nodes from a patient, and expanding the lymphocytes in vitro. An independent claim is also included for kit for carrying out (M1) comprising a dye and a substance capable to stimulate proliferation of lymphocytes. ACTIVITY : Cytostatic. Five patients with colon cancer, with no signs of distant metastases or lymph node involvement prior to surgery, were included in the study. The colonic tumor site was mobilized through division of peritoneal adhesions to facilitate inspection. One ml Patent blue dye was injected superficially in the circumference of the tumor. Within five minutes, one to three blue-colored mesenteric lymph nodes were identified macroscopically as sentinel nodes and they were marked with sutures. The sentinel and non-sentinel nodes were cut in half. Slices less than 1 mm thick were cut from the central and the peripheral part of the nodes for flow cytometry and proliferation analysis. The rest of the node underwent routine histopathological examination. Tumors were histopathologically classified as Dukes stages A-C. Upon macroscopical dissection of the removed specimens, between 18 and 29 lymph nodes were identified and embedded for histopathological evaluation. Patients 2 and 3 had metastatic spread to the sentinel node and were histopathologically classified as Dukes C. The other 3 patients did not show any signs of metastatic spread to sentinel node(s) nor to other lymph nodes despite tumors grew through the bowel muscular wall. They were classified as Dukes B. Single cell suspensions of lymphocytes collected separately from peripheral blood (PBL), the tumor, draining sentinel lymph nodes and non-draining lymph nodes were tripe stained with antibodies recognizing the very early activation marker CD69 and the T-cell markers CD4 and CD8 followed by flow cytometry analyzes (FACS). Similar numbers of activated CD4+ lymphocytes, where found both in sentinel and non-sentinel nodes regardless of presence or absence of metastases. All investigated tumors contained tumor infiltrating lymphocytes (TILs) to a various extent and the majority of these TILs, which were both CD4+ AND CD8+ T-cells, presented and activated CD69+ phenotype. In peripheral blood activated CD4+CD69+ lymphocytes were absent. The functional status of the lymphocytes was further characterized in time course proliferation assays using homogenized tumor cell extracts as antigen source. Stimulation with Patent blue dye did not cause proliferation in any case. In all sentinel lymph nodes without metastasis (Dukes B cases), the lymphocytes proliferated in a dose dependent manner against autologous tumor extract. Peak proliferation was regularly seen at day 6 at the highest amount of antigen. No antigen dependent proliferation was recognized among lymphocytes from non-sentinel nodes or from TILs. In the Dukes C cases, where the sentinel nodes contained metastatic cells, none or a very weak proliferative response was identified. To further investigate the functional state of the lymphocytes, T-cells were non-specifically stimulated with Concanavalin A which by-pass the activation through the T-cell receptor. All PBLs and non-sentinel node lymphocytes responded with strong proliferation as well as sentinel node lymphocytes from the Dukes B patients. However, sentinel node lymphocytes from the Dukes C patients did not respond with proliferation against Concanavalin A, nor did the TILs from Duke B or C cases. Stimulation of sentinel node lymphocytes from a Dukes B patient with 100 microg/ml of carcinoembryonic antigen did not result in any proliferative activity. Lymphocytes with specific activity towards autologous tumor extract were selected and put in long term cultures. The stimulated cells consisted of predominantly CD4+ T lymphocytes and they were expanded in the presence of interleukin (IL)-2 and survived in vitro for several weeks. MECHANISM OF ACTION : Immunotherapy. No supporting data is given.